High throughput screening of G-protein coupled receptors via flow cytometry

The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.     

Virtual high throughput screening in new lead identification

Drug discovery continues to be one of the greatest contemporary challenges and rational application of modelling approaches is the first important step to obtain lead compounds, which can be optimised further. Virtual high throughput screening (VHTS) is one of the efficient approaches to obtain lead structures for a given target. Strategic application of different screening filters like pharmacophore mapping, shape-based, ligand-based, molecular similarity etc., in combination with other drug design protocols provide invaluable insights in lead identification and optimization. Screening of large databases using these computational methods provides potential lead compounds, thus triggering a meaningful interplay between computations and experiments. In this review, we present a critical account on the relevance of molecular modelling approaches in general, lead optimization and virtual screening methods in particular for new lead identification. The importance of developing reliable scoring functions for non-bonded interactions has been highlighted, as it is an extremely important measure for the reliability of scoring function. The lead optimization and new lead design has also been illustrated with examples. The importance of employing a combination of general and target specific screening protocols has also been highlighted.     

Solution phase enantioselective recognition and discrimination by electrospray ionization--mass spectrometry: state-of-the-art, methods, and an eye towards increased throughput measurements

A review is given from the stand-point of applying methods utilizing electrospray ionization - mass spectrometry (ESI-MS) for quantitative binding (affinity and selectivity) determinations in small molecule host-guest (receptor-ligand, selector-selectand, etc.) systems. Advantages over more commonly utilized and traditional solution phase approaches, both in the context of developing new chiral selector molecules for separation and purification of enantiomers and in drug discovery applications, are presented. Although the majority of studies employing methods such as host-guest screening, competitive binding, and titrations have focused on large protein-ligand, DNA-ligand, and RNA-ligand systems, the use of ESI-MS for studying small molecule and chiral recognition systems is growing. For the latter case, greater care must be given to considering the effects of the ESI process on the ionization of the species involved in equilibria of interest. Some basic mechanistic and practical concerns for performing solution-phase-targeting quantitative measurements by ESI-MS are given in this light. Finally, an account of the application of these methods in a high throughput format is given, highlighting the potential of traditional and novel screening and titration approaches which allow scientists to screen the performance of hundreds, if not thousands, of compounds in a single day.     

The multi-copy simultaneous search methodology: a fundamental tool for structure-based drug design

Fragment-based ligand design approaches, such as the multi-copy simultaneous search (MCSS) methodology, have proven to be useful tools in the search for novel therapeutic compounds that bind pre-specified targets of known structure. MCSS offers a variety of advantages over more traditional high-throughput screening methods, and has been applied successfully to challenging targets. The methodology is quite general and can be used to construct functionality maps for proteins, DNA, and RNA. In this review, we describe the main aspects of the MCSS method and outline the general use of the methodology as a fundamental tool to guide the design of de novo lead compounds. We focus our discussion on the evaluation of MCSS results and the incorporation of protein flexibility into the methodology. In addition, we demonstrate on several specific examples how the information arising from the MCSS functionality maps has been successfully used to predict ligand binding to protein targets and RNA.     

A neural network based virtual high throughput screening test for the prediction of CNS activity

A virtual high throughput screening test to identify potentially CNS-active drugs has been developed. Discrimination was based on the knowledge available in databases containing CNS-active (Cipsline from Prous Science) and inactive compounds (Chemical Directory from Sigma-Aldrich). Molecular structures were represented using 2D Unit y fingerprints and a feedforward neural network was trained to classify molecules regarding their CNS activity. The parameterized network was validated by reclassification of the training set elements, by the classification of a test set preselected from the Prous database, and also by the prediction of activity for known CNS drugs not used in the training set but available in the Medchem database (Daylight). These tests revealed that our neural net recognized at least 89% of CNS-active compounds and would be suitable for use in our virtual screening protocol.     

Peptide display in functional genomics

The completion of the human genome project has opened novel scientific avenues in functional genomics, structural genomics and proteomics. These areas have a common goal: the identification of all the proteins acting and cross-talking in a single cell at a defined moment of its lifecycle. The expansion of these areas in bioscience has been facilitated by the rapid development of high throughput screening (HTS) methods which has, in turn, attracted the business community to make investments in this novel business segment of biotechnology. By using these HTS methods, the hope is that novel targets will be validated much more rapidly speeding up the development of novel drugs. Numerous techniques and tools have emerged over the past decade for the identification of small target-specific molecular ligands that exploit a common feature: the exploration of molecular diversity using combinatorial methods. While chemists developed new methods for rapidly and efficiently synthesising and screening large collections of small molecules, biologists used recombinant DNA techniques for selecting displayed repertoires. To this end, the discovery of new low molecular weight peptides is becoming increasingly important, not only as molecular tools for the understanding of protein-protein interactions but also for the generation of lead compounds.     

Acoustic deposition with NIMS as a high-throughput enzyme activity assay

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.     

The application of high throughput siRNA screening technology to study host-pathogen interactions

Recent advances in high throughput screening technologies have accelerated the identification and characterization of potential factors involved in host-virus interactions, facilitating early detection and diagnosis of diseases, as well as providing promising drug targets. The last decade has seen a plethora of successful examples of high throughput screening approaches, especially siRNA screening. With support from protein interaction studies, mRNA expression profiling, and bioinformatics, siRNA screening has also been successfully utilized to identify host factors required for a number of viruses including HIV, West Nile virus and H1N1 virus. Such studies have raised the awareness of virologists, and have opened a new chapter of global analysis of host-pathogen interactions. However, to play a more defining role in prognostics, diagnostics and therapeutics for virus diseases, acknowledged drawbacks, including false positives and negatives, inherent in this technology, must be successfully addressed.     

Structural genomics on membrane proteins: mini review

Structural genomics, structure-based analysis of gene products, has so far mainly concentrated on soluble proteins because of their less demanding requirements for overexpression, purification and crystallisation compared to membrane proteins. This so-called "low-hanging fruit" approach has generated more than 25,000 structures deposited in databases. In contrast, the substantially more complex membrane proteins, in relation to all steps from overexpression to high-resolution structure determination, represent less than 1% of available crystal structures. This is in sharp contrast to the importance of this type of proteins, particularly G protein-coupled receptors (GPCRs), as today 60-70% of the current drug targets are based on membrane proteins. The key to improved success with membrane protein structural elucidation is technology development. The most efficient approach constitutes parallel studies on a large number of targets and evaluation of various systems for expression. Next, high throughput format solubilisation and refolding screening methods for a wide range of detergents and additives in numerous concentrations should be established. Today, several networks dealing with structural genomics approaches of membrane proteins have been initiated, among them the Membrane Protein Network (MePNet) programme that deals with the pharmaceutically important mammalian GPCRs. In MePNet, three overexpression systems have been employed for the evaluation of 101 GPCRs, which has generated large quantities of numerous recombinant GPCRs, compatible for structural biology applications.     

Ion channel screening

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts. In addition, major advances have been made in the development of automated electrophysiological platforms to increase capacity for cell-based screening using formats aimed at recapitulating the gold standard assay. This review addresses the options available for cell-based screening of ion channels with examples of their utility and presents case studies on the successful implementation of high-throughput screening campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye.     

Bio- and chemiluminescence in bioanalysis

Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio- and chemiluminescent detection make it suitable for the development of microarray-based high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.     

Microfluidics for cell-based high throughput screening platforms—A review

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.     

Development of Enantioselective Fluorescent Sensors for Chiral Recognition

Novel chiral compounds have been synthesized for the enantioselective fluorescent recognition of alpha-hydroxycarboxylic acids and amino acids. By introducing dendritic branches to the chiral receptor units, the fluorescence signals of the receptors are significantly amplified because of the light-harvesting effect of the dendritic structure. This has greatly increased the sensitivity of the sensors in the fluorescent recognition. Highly enantioselective fluorescent responses have also been ac…     

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.     

High throughput screening technology and the small molecules modulating aging related signals

Aging and its related diseases are severe issues in modern society. Many efforts have been made to understand the mechanisms of aging and to find the ways to prevent age-related diseases. Identifying the compounds targeting aging-related signals is a challenging work because there are so many proteins and signals involved. Recently, alone with the progresses in high throughput screening (HTS) technology, increasing numbers of small molecules targeting aging-related pathologic processes have been identified. In this review, we introduce the basic workflow, classification and assay strategies of HTS technology, and sort out known small molecules identified via HTS technology by their roles in aging related diseases, such as neural degenerative diseases, diabetes and tumors. Given the fact that application of HTS on aging research is still at an early stage, we also summarize the cellular mechanisms about aging process, paralleled with the compounds which can modulate the functions of proteins important for aging signals. Finally, we briefly discuss some advanced HTS technologies for their potent applications on the discovery of anti-aging compounds. The main purpose of this review is to provide updated and useful information to those who are interested in pharmacology and HTS technology, but not familiar with aging biology, or vice versa.     

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 μm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P _oct measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as β-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-μm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application. Figure The UHPLC approach described here greatly enhanced the time required for log P determination (5' min by compound using UV detection) and, at least, 8 compounds measured in a 5' run when Mass Spectrometry detection in used. These developments offer to medicinal chemists a high-throughput method to estimate the lipophilicity of NCEs Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Experimental section for capillary electrophoresis (CE) measurements, list of the 38 compounds of the calibration set and solvatochromic analyses of the extrapolated retention factors and partition coefficients.     

Evaluation of a method for high throughput solubility determination using a multi-wavelength UV plate reader

Aqueous solubility is a critical physicochemical property and must be addressed early during drug discovery research. Due to the difficulty in accurately predicting aqueous solubility in silico, high throughput experimental determination of aqueous solubility is in great demand. This study evaluates a method using a multi-wavelength UV plate reader and disposable 96-well UV plates for fast solubility determination. It was demonstrated that this method has the sensitivity and reproducibility to effectively determine solubility as low as 1 micro M. Excellent correlation (R>0.97) was observed between the solubility determined using the UV reader method and the HPLC method over the range of 1-1000 micro M for a diverse set of pharmaceutical compounds. In addition to excellent sensitivity and reproducibility, the UV plate reader method also offers the flexibility of being able to determine thermodynamic solubility in the presence or absence of dimethyl sulfoxide, which is a solvent widely used for combinatorial compounds during high throughput screening.     

Targeting chemical inputs and optimising HTS for agrochemical discovery

In vivo high throughput screening (HTS) has been adopted by most of the larger crop protection companies as an important tool for the discovery of new agrochemicals. There has been a paradigm shift in capabilities from screening a few thousand compounds a year to several hundred thousand and the quantity of screening sample required has fallen dramatically. The unifying goal now bringing together screens and inputs is the need to maximise the flow of useful information from HTS and thereby minimise the time taken to discover robust leads and new products. This review examines the positive changes that have occurred towards targeted design and selection of chemical inputs for agrochemical discovery over the last ten years and corresponding developments in HTS assays, data analysis and the logistics of compound storage and dispensing.     

临近闪烁分析法在高通量筛选中的应用

起源于放射性免疫分析的临近闪烁分析法(scintillation proximity assay,SPA)是一种均相、灵敏、快速和简便的基于闪烁载体的分析平台。该平台可用于筛选药物靶点的先导化合物和研究其生理过程。由于无需分离,易于固定药物靶点和检测其活性,SPA成为一种重要的高通量筛选方法。由于放射性标记分子和亲和标签分子的多样化和商业化、以及液闪计数器和液相操作等技术的发展,SPA已经广泛用于受体结合、高通量药物筛选、酶分析、放射性免疫分析、蛋白质-蛋白质相互作用和细胞水平分析等方面。本文阐述了SPA原理,讨论了其关键技术(包括闪烁载体、液闪计数器和放射性标记分子),分析了其评价体系;同时简述了SPA分析的发展, 并介绍了其在高通量筛选中的应用实例, 归纳了存在的问题,给出了未来的发展趋势。目前,基于SPA和荧光分析方法已成为高通量药物筛选的热点研究领域, 这些筛选技术的革新必然提升我们对细胞体系生物学的全面理解和促进先导化合物筛选过程的显著进步。